Is Transcriptomic Regulation of Berry Development More Important at Night than During the Day?

Is Transcriptomic Regulation of Berry Development MoreImportant at Night than During the Day?Markus Rienth1,2, Laurent Torregrosa2, Mary T. Kelly3, Nathalie Luchaire2,4, Anne Pellegrino4,

Jerome Grimplet5, Charles Romieu6*

1 Fondation Jean Poupelain, Javrezac, France, 2 INRA-SupAgro, UMR AGAP, Montpellier, France, 3 Laboratoire d’Oenologie, UMR1083, Faculte de Pharmacie, Montpellier,

France, 4 INRA, UMR LEPSE, Montpellier, France, 5 ICVV (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Logrono, Spain, 6 INRA, UMR AGAP, Montpellier, France

Abstract

Diurnal changes in gene expression occur in all living organisms and have been studied on model plants such as Arabidopsisthaliana. To our knowledge the impact of the nycthemeral cycle on the genetic program of fleshly fruit development hasbeen hitherto overlooked. In order to circumvent environmental changes throughout fruit development, young andripening berries were sampled simultaneously on continuously flowering microvines acclimated to controlled circadian lightand temperature changes. Gene expression profiles along fruit development were monitored during both day and nightwith whole genome microarrays (NimblegenH vitis 12x), yielding a total number of 9273 developmentally modulatedprobesets. All day-detected transcripts were modulated at night, whereas 1843 genes were night-specific. Very similardevelopmental patterns of gene expression were observed using independent hierarchical clustering of day and night data,whereas functional categories of allocated transcripts varied according to time of day. Many transcripts within pathways,known to be up-regulated during ripening, in particular those linked to secondary metabolism exhibited a clearerdevelopmental regulation at night than during the day. Functional enrichment analysis also indicated that diurnallymodulated genes considerably varied during fruit development, with a shift from cellular organization and photosynthesisin green berries to secondary metabolism and stress-related genes in ripening berries. These results reveal critical changesin gene expression during night development that differ from daytime development, which have not been observed inother transcriptomic studies on fruit development thus far.

Citation: Rienth M, Torregrosa L, Kelly MT, Luchaire N, Pellegrino A, et al. (2014) Is Transcriptomic Regulation of Berry Development More Important at Night thanDuring the Day? PLoS ONE 9(2): e88844. doi:10.1371/journal.pone.0088844

Editor: Nicholas S. Foulkes, Karlsruhe Institute of Technology, Germany

Received October 4, 2013; Accepted January 12, 2014; Published February 13, 2014

Copyright: � 2014 Rienth et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work is part of the DURAVITIS program which is financially supported by the ANR (Agence national de la recherche) -Genopole (project ANR-2010-GENM-004-01) and the Jean Poupelain foundation (30 Rue Gate Chien, 16100 Javrezac, France). The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

The grapevine is one of the most abundant perennial crops in

the world with a total surface of approximately 7.6 million hectares

planted under vines [1]. Complex, poorly understood processes,

occurring at different stages throughout berry development

determine the final quality of the fruit. The development of the

grapevine berry follows a double sigmoid growth pattern

consisting of two distinct growth phases separated by a lag phase

[2]. Cell division triggered at anthesis occurs only during the first

phase of berry development, which lasts approximately 50 to 60

days after flowering, depending on cultivar and environmental

conditions [3,4]. This stage is marked by a first period of vacuolar

expansion that relies on the synthesis and storage of tartaric and

malic acid [5] as the major osmoticums at a vacuolar pH of

approximately 2.6 [6]. Several other compounds, with an

important effect on ultimate wine quality are also accumulated

during the first growth period of the berry. Amongst these are

hydrocinnamic acids, tannins, amino acids [7,8,9] and some

aroma compounds such as methoxypyrazines in varietals such as

Cabernet Sauvignon, Cabernet Frank and Sauvignon blanc

[10,11]. The first growth phase is followed by a lag phase where

berry growth and organic acid accumulation cease. The most

significant changes in gene expression are triggered during the

24 h transition phase between the lag phase and ripening, where

berries suddenly soften individually [12,13]. During the subse-

quent ripening phase, the volume of the berry roughly doubles,

with the accumulation of approximately 1 M hexoses as

osmoticums, and the respiration of malic acid is induced

simultaneously with sugar loading. During ripening, amino acids

and anthocyanins accumulate [3] and major aromatic compounds

including terpenes, norisoprenoids, esters and thiols are synthe-

sized [10]. The control of these physiological processes is not well

understood in the grapevine – which is a non-climacteric fruit

exhibiting completely different developmental characteristics from

climacteric fruit such as tomato or banana which have been more

extensively studied [14].

Since the publication of the grapevine Genome in 2007 [15]

several high-throughput technologies have been developed in

order to gain a greater understanding of the regulation of

physiological changes occurring during berry development.

Studies using microarrays or RNA sequencing technology have

been carried out on economically important Vitis Vinifera L.

cultivars, for example Chardonnay, Muscat de Hamburg,

Trincadeira, Cabernet Sauvignon, Shiraz, Corvina and Pinot

PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e88844

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Noir. [13,16,17,18,19,20,21,22]. These studies led to a greater

understanding of some traits of berry ripening including the

regulation of tannin and anthocyanin biosynthesis pathways [23].

However, major physiological events such as the onset of malic

acid respiration are not fully understood at this time [24,25].

Presumably the lack of significant transcriptional changes in such

studies is due to sampling protocols that did not pay sufficient

attention to specific time points during berry development. Other

possible reasons are uncontrolled environmental conditions

leading to the introduction of significant, unquantifiable biases in

gene expression, covering developmentally regulated changes.

All studies on grapevine berry development have been

conducted on field grown grapevines where impacts on gene

expression arising from environmental conditions cannot be

avoided. Furthermore, all studies on berries and other fleshy

fruits were carried out during the day. For this reason changes

occurring throughout berry development during the night were

neglected, despite the knowledge of significant diurnal changes,

such as fruit swelling during the nighttime [26,27], daytime-

dependent regulation of photosynthesis [28] and changes in gene

expression related to the circadian clocks. The latter, whose

central function is to sustain robust cycling across a wide range of

light and temperature conditions are known to regulate physiology

in order to respond to the day/night cycle [29]. Circadian timing

involves the rhythmic expression of genes that were identified in

many organisms and tissues from cyanobacteria to mammals

[30,31]. Studies of gene expression by transcriptomics were the

first global experiments to provide information on the molecular

rhythms at the whole plant level [32]. Early time–course studies

estimated that 2–16% of the steady state transcriptome is

regulated by the circadian clock with peak phases occurring

throughout the day [33,34]. The circadian effect is well buffered

across a range of temperatures and conditions by a compensatory

mechanism [35]. This is the first study where gene expression

during berry/fleshy fruit development was characterized simulta-

neously during the day and at night.

The studied microvine is a GAI1 (GA insensitive) mutant

regenerated from the L1 cell layer of Pinot Meunier L., exhibiting

a dwarf stature and an early and continuous fructification along

the main vegetative axis [36,37]. It was previously proposed as a

new model for grapevine research in genetics and physiology

[38,39,40] and was shown to be adapted for small scale

experiments in climatic chambers [41]. The dwarf stature of the

microvine made it possible to grow plants under strictly controlled

conditions during the whole period of reproductive development,

and to obtain simultaneously, on the same plant, fruits at different

developmental stages, thus minimizing the introduction of

environmental biases linked to field conditions or noticeable

changes in photoperiod during the reproductive cycle. A whole

genome approach with Vitis 12X NimblegenH 30 K microarrays

was used on four different developmental stages sampled during

the day and night. Results show that developmental regulation of

gene expression at night is very critical for grapevine fruit

development with many genes responding in a different manner

between developmental stages. The number and categories of

modulated genes between day and night differ tremendously

depending on the different stages of berry development especially

between the green and the ripening berry.

Results and Discussion

Stage Selection and Validation of Experimental DesignBerries at six developmental stages were sampled simultaneously

during the day or night: berry set (BS), two stages during green

growth (G1, G2), lag phase or ‘‘plateau herbace’’ (PH) and two

ripening phases (R1 and R2; Figure 1). Berries from microvines

displayed the same three typical phases of development as field

vines in relation to the evolution of fresh weight and major solutes

(Figure 1). The first or green growth period where malic acid

concentration increases up to 280 mEq is followed by the lag

phase with berry growth and acid accumulation leveling off at

around 0.6 g berry weight. Thereafter growth is resumed; hexose

accumulation starts simultaneously with the breakdown of malic

acid, until berry weight reaches 1.4 g and hexoses reach 1 M at

maturity. Tartaric acid accumulation ceases at 120 mEq during

the first growth period, yielding a malate to tartrate ratio of 2.3,

before reducing in concentration due to dilution, while remaining

constant on a per berry basis (data not shown).

The amino acid profile of berries is presented in Table S1. The

most abundant amino acids of the microvine berry were proline

(pro), arginine (arg) followed by alanine (ala), glutamic acid (glu),

aspargine (asp), threonine (thr), glutamine (gln) and lysine. Free

amino acid concentrations vary depending on cultivar, rootstock/

scion combinations, vine nutrient management, vineyard site, and

growing season [43]. However, the microvine presents an amino

acid profile comparable to field grapevine cultivars [42,43]. From

these observations, it can be concluded that the gai1 mutation in

the dwarf phenotype of the microvine does not impact major fruit

developmental features. This can be explained by the tissue

specificity of GAI1 that is expressed in several grapevine organs but

not fruits, conversely to other GAIs genes (data not shown).

Four stages were selected for transcriptomic analysis, including

two stages in each successive growth period. Berry growth and

acid accumulation occurred at maximal rate in G1 and more

slowly in G2, just before the lag phase. In the same manner, two

stages were selected during ripening, which share quite close

physiological characteristics, but with slower growth and sugar

import rates in R2 as compared to R1.

Of the 9273 transcripts detected as modulated between at least

two stages, (fold change (fc) .2; pval adj ,0.05), 7430 of these

were simultaneously detected in both day and night samples; 1843

appeared in the night only, whereas none were restricted to day

samples (Table S3). This repartition a posteriori validates robust

changes in gene expression hitherto obtained through day-

screenings as reported in the literature

[13,16,17,18,19,20,21,22]. However, a substantial part of devel-

opmentally regulated changes in gene expression occurring

specifically at night was totally overlooked so far. Transcripts

modulated in microvine berries between green and ripening stages

were compared with data extracted from Fasoli et al., 2012 [44]

conducted on Vitis vinifera cv Corvina berries, available in the Gene

Expression Omnibus under the series entry GSE36128 (http://

www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token = lfcrxesyciqgs

joandacc = GSE36128).

1970 transcripts were detected in Corvina berries between the

stages called ‘‘post-fruit set’’ (green berry) and ‘‘ripening’’. Of

these, 1550 (79%) were also modulated in microvine between

green and ripening berries (Table S6) and showed good linear

correlation in their expression (R2 = 0.72; Figure S5). The large

number of commonly modulated genes despite different geno-

types, environmental conditions and sampling stages, validates the

microvine as a model for the study of berry physiology and

transcriptomics. In contrast, it must be emphasized that 94% of

the 1843 genes detected here that were specifically modulated

during nighttime development have not been observed in daytime

experiments on Corvina berries.

Analysis of the data at each of the four stages revealed that 2684

transcripts changed expression during the day/night transition at

Day - Night Transcriptomics of Berry Development


http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?tokenfcrxesyciqgs

http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?tokenfcrxesyciqgs

one developmental stage at least. Amongst them 1849 (70%) also

showed developmental changes between individual growth stages.

An overview of down- and up-regulated transcripts between day

and night is presented in Table S2. Principal component analysis

(PCA; Figure 2) was applied separately on the two green stages, the

two ripening stages and between G1 and R1. The two green stages

are separated by the first PC explaining half of the variation in

gene expression with greater differences for the night samples

compared to day samples. The second PC, accounting for 11% of

the variation in gene expression represents the day/night axis and

shows a clearer separation for G2. The PCA on ripening stages

yields an inversion of these axes, with PC1 explaining once again

half of the variation but separating day and night, while

developmental stages can be distinguished by PC2 (14% variance)

for the night samples only. In the plot between G1 and R1 90%

variance can be attributed to development (PC1) and only 4%

account for day and night differences (PC2). This large variation

between green and ripe berries concurs with the fact that most

important changes in gene expression occur at the onset of

ripening in developing berries [12,13]. The day/night discrimi-

nation explained by PC2 is more pronounced for the later rather

than for the earlier developmental stages. These results highlight

the importance of considering the berry transcriptome at night

where close stages seem to show more significant differences than

during the day.

Developmentally Regulated Gene ExpressionThe previous 9273 developmentally regulated transcripts were

allocated to the same number of clusters, treating day and night

samples separately. Both independent hierarchical clusterings

yielded very similar expression patterns for day and night

(Figure 3), however, a large number of transcripts differed between

day and night in corresponding clusters. Functional categories

over-represented in each cluster were obtained through enrich-

ment analysis (Figures S1 to S4). Transcripts induced during

ripening (cluster 1) only during the day or at night are illustrated in

Figure 4A together with those repressed during ripening (cluster 2;

Figure 4 B). This highlights developmentally regulated processes

Figure 1. Main biochemical characteristics of sampled berries. BS: Berry Set, G1: Green stage 1, G2: Green stage 2, PH: Plateau Herbace/lagphase, R1: Ripening stage 1, R2: Ripening stage 2.doi:10.1371/journal.pone.0088844.g001



and their diurnal dependence. Flavonoid metabolism, amino acid

metabolism and cell wall-related processes were noticeably

induced in ripening berries during the night and not specifically

during the day. A large number of photosynthesis-related genes

were repressed only at night between young and ripening stages.

This highlights the need to include nighttime sampling in

developmental studies in order to investigate a substantially wider

range of transcriptomic changes.

Day/Night Modulated TranscriptsA second approach consisted of screening for genes differentially

expressed between day and night at all four developmental stages

(Figure 5). Surprisingly, very few transcripts (3 and 6) remained

day/night modulated throughout berry development. This indi-

cates that no pure mechanism of diurnal regulation prevails over

all developmental stages. Many day/night-modulated genes were

actually conserved within the green or the ripening group. In this

respect, most genes in green berries were modulated between day

and night in G2, whereas the differences in ripening berries were

not as obvious. Berries at the end of the first growth period (G2)

seem consequently to be most responsive to diurnal changes when

compared to other stages. Functional classes of transcripts down-

or up-regulated during the night were clearly separated between

young and ripening berries (Figure 6). Modulated genes in young

berries are mainly attributed to cellular division/expansion events

that occur during the green growth phase (cell growth, cellulose

catabolism, xyloglucan modification, microtubule-driven move-

ment, oil entity organization). At green stages, the berry exhibits

marked diurnal changes in volume consisting of night expansion

followed by day contraction due to berry transpiration and water

backflow to the canopy through xylem vessels [25,49]. This large

amplitude in cell expansion triggered at night places an additional

demand on cell wall structural components. In ripening berries cell

division has ceased and the diurnal pattern of swelling is strongly

reduced by the impairment of xylem conductance preventing

water backflow [25]. Consequently cellular growth-related cate-

gories are no longer significantly enriched within day/night-

modulated transcripts. Photosynthesis (PS)- associated transcripts

are repressed at night in the green berry, which may be due to the

lack of light reactions of the PS system. In the ripening berry,

diurnal changes of gene expression occur mainly within secondary

metabolism, whereas categories like phenylpropanoid, terpenoid

and stilbene biosynthesis were enriched in night-induced tran-

scripts. Interestingly, genes within the latter category inverse their

diurnal pattern between green and ripening berries. A switch from

symplastic to apoplastic phloem unloading is known to occur in

ripening berries [45], with hexoses (mainly fructose and glucose)

being stocked in the vacuoles. Once ripening has started the berry

has thus its own sugar reserves, which can be used for the synthesis

of secondary metabolites.

Indications of Oxidative Burst Occurring at Night inRipening Berries

Oxidative burst is known to occur during ripening of climacteric

fruit, but some studies have indicated that this phenomenon can

also take place in non-climacteric fruit such as the grapevine

[13,46,47]. Overexpression of genes involved in ROS scavenging

peaking immediately after the onset of ripening was observed by

several authors [17,48], but its regulation at the transcriptional

level remains unclear since these stress markers seemed to be

absent in other studies [12]. Remarkably and what has never been

previously observed, is that oxidative stress seems to occur in

ripening berries at night, where functional categories related to

oxidative stress response were enriched in up-regulated transcripts

(Figure 6). This observation is confirmed by the fact that genes of

the RBOH (respiratory burst oxidase protein) family

(VIT_14s0060g02320, VIT_01s0150g00440 and

VIT_02s0025g00510) are concomitantly induced at night in

ripening berries (Table S4). RBOHs encode the key enzymatic

subunit of plant NADPH oxidase and support the production of

ROI (reactive oxygen intermediates) following biotic and abiotic

stresses in plants [49]. Ascorbate oxidase isogenes

(VIT_07s0031g01040, VIT_07s0031g01120, VIT_07s0031g01120)

were also induced at night in R2 (Table S4). This family of ROI

scavenging enzymes has been associated with the control of cell

growth and the stress response [50]. A large number of peroxidase

and laccase coding transcripts were found to be up-regulated in

ripening berries at night (Table S4) in agreement with the night

stress hypothesis. Ectopic expression of laccase in yeast confers

improved H2O2 scavenging activity and hereby protect cells from

lipid oxidative damage upon stress [51]. An up-regulation of

RBOH could also be attributed to cell elongation at night during

Figure 2. Principal component analysis separately on green stages (left), ripe stages (right) and between green and ripe (middle)during the day and night.doi:10.1371/journal.pone.0088844.g002



ripening. Studies on Arabidopsis thaliana RBOHc (Atrbohc) mutants

indicated that ROIs activate hyper-polarization Ca2+ channels

which are responsible for localized cell expansion during root-hair

formation [52]. The induction of a calcium-transporting ATPase

Figure 3. Expression profiles of developmentally regulated genes during the day and night. Clustering was performed using k-meansstatistics on mean centered RMA normalized expression log2 values. Numbers of all day respectively night specific transcripts in each cluster aredisplayed.doi:10.1371/journal.pone.0088844.g003



coding transcript (VIT_13s0158g00360) concomitant with calmod-

ulin-binding proteins, and a calcium/proton exchanger (CAX 3;

VIT_08s0007g02240; Table S4) may indicate day/night changes

in the homeostasis of cytosolic Ca2+ in ripening berries. A

cessation of Ca2+ importation actually results from the marked

shift from xylem to phloem conductance at the onset of ripening

[53]. In plants, stress initiates a signal-transduction pathway, in

which the synthesis of c-aminobutyric acid is increased [54]. This

Figure 4. Example of genes allocated to illustrated clusters (4A: cluster 1 and 4B: cluster 2) specifically during day (red) or night(blue). Scales are log2 values calculated between G2 and R1.doi:10.1371/journal.pone.0088844.g004

Figure 5. Overview of day/night modulated transcripts (fold change .2; pval adj ,0.05) in each developmental stage. Left diagramnight down-regulated transcripts; Right diagram night up-regulated transcripts.doi:10.1371/journal.pone.0088844.g005



amino acid transiently accumulates in anoxic ripe berries and is

rapidly re-oxidized upon restitution of air supply [55]. The up-

regulation at night of a c-aminobutyric acid transporter

(VIT_13s0074g00570; Table S4) suggests that glutamate decar-

boxylase [56] and GABA shunt activities may be day/night

modulated by changes in cytosolic Ca2+ (see above), pH, or redox

state [57] in ripening berries. Furthermore, the transcription factor

(TF) family WRKY was over-represented in R1 at night (Figure 6).

TFs of the latter family were shown to respond to various types of

biotic stress in rice [58].

Since growth in the ripening berry is due only to cellular

expansion, the data suggests that this occurs mainly during the

night. Additionally it supports the hypothesis presented in

following section on carbohydrates that sugar importation into

the ripening berry may principally occur during the night. It may

also be hypothesized that sugar uploading into the vacuole

increases osmotic pressure and thus represents a stressor for the

cells.

Carbohydrate Transport Related Transcripts Inverse theirDay/Night Modulation from Green to Ripe Berries

Matching the pattern of sugar accumulation, sugar transporters

(ST; VIT_14s0006g03290, VIT_14s0083g00010,

VIT_14s0083g00020, VIT_14s0083g00030, VIT_05s0020g03140)

were up-regulated in ripening berries (cluster 1 day and night;

Table S3 and S5) concomitantly with hexose transporters (HT1

and HT7; VIT_16s0013g01950, VIT_11s0149g00050; Table S4).

Curiously, all detected ST transcripts showed night up-regulation

in the R2 (Table S4). This suggests that the apoplasmic pathway of

sugar loading may be activated during the night with starch

accumulation in chloroplasts occurring during the day and

subsequent translocation as phloem-mobile sucrose during the

night.

Interestingly sucrose synthase transcripts (SuSy;

VIT_05s0077g01930, VIT_10s0071g00070, VIT_00s1562g00010,

VIT_11s0065g01130, VIT_12s0057g00130) were induced during

the night in green berries before the lag phase (Table S4).

Frequently associated with sink tissues, SuSy are thought to be

cytoplasmic enzymes in plant cells where they serve to degrade or

synthesize sucrose and provide carbon for respiration and UDP-

glucose for the synthesis of cell wall polysaccharides and starch

[59,60,61]. It has also been reported that SuSy are tightly

associated with the plasma membrane and therefore might serve

to channel carbon directly from sucrose to cellulose and/or callose

synthases in the plasma membrane [62]. This indicates that

assimilated sugar is processed to cell wall compounds important

for cell development in the night in green berries. Presented

hypothesis is discussed in more detail in the section regarding cell

division.

Principal Events in the Phenylpropanoid Pathway Seemto be Regulated at Night during Ripening

Phenolic compounds are important substances determining

wine quality; they are partly responsible for color and astringency,

and at the same time for numerous physiological benefits

associated with moderate wine consumption [63]. Most phenolics

derive from the non-oxidative deamination of phenylalanine via

phenylalanine-ammonia-lyase (PAL) and encompass a range of

structural classes such as lignins, phenolic acids, flavonoids and

stilbenes [64]. Significant parts of the phenylpropanoid pathway

and the day/night modulation of its enzymes are illustrated in

Figure 7. A large number of isogenes within this pathway were

repressed during the day (in relation to up-regulated at night)

specifically at the ripe stages. In particular, almost all transcripts

coding for the key enzyme PAL were up-regulated at night in ripe

berries, signifying that major secondary processes take place

during this final phase of development. Accordingly, transcripts

coding the enzymes hydroxycinnamoyl-CoA shikimate/quinate

hydroxycinnamoyltransferase (VIT_11s0037g00440) and p-coumar-

oyl shikimate 3’-hydroxylase (VIT_08s0040g00780), important ele-

ments of the shikimic acid pathway, were concomitantly modu-

lated at night in ripening berries (Table S4). The shikimic acid

pathway converts simple carbohydrate precursors derived from

glycolysis and the pentose phosphate pathway to the aromatic

amino acids tyrosine and phenylalanine, and thus provides the

latter for the phenylpropanoid pathway [65]. Most transcripts

coding for tri-hydroxy-stilbene-synthase, inversed their day/night

modulation between the green and ripening stages (Figure 7) - they

exhibited induction during night in ripening berries and vice versa

in green berries. This implies that stilbene synthesis in ripening

berries takes place during the night and vice versa during green

growth stages, which is supported by the fact that resveratrol synthases

(RS; VIT_16s0100g01110, VIT_16s0100g01070) are concomitantly

regulated. RS intervenes in the final synthetic step of resveratrol,

an important phytoalexin that has been shown to possess

antioxidant and anti-inflammatory properties [66,67].

Proanthocyanidin (PA) biosynthesis is part of the phenylpropa-

noid pathway that also produces anthocyanins and flavonols. PAs

are polymers of flavan-3-ol subunits and often referred to as

condensed tannins. They protect plants against herbivores, are

important quality components of many fruits and constitute the

majority of wine phenolics [68]. Two enzymes, leucoanthocyanidin

reductase (LAR) and anthocyanidin reductase [69] can produce the

flavan-3-ol monomers required for formation of PA polymers

[70,71]. Transcripts coding for ANR (VIT_00s0361g00040) and

LAR (VIT_17s0000g04150, VIT_01s0011g02960) were consistently

down-regulated throughout berry development (cluster 7; Table

S5). The expression of the second LAR transcript in young green

berries was twice as pronounced during the night as during the day

throughout development (Table S3), underlining the importance

of studying gene expression profiles at night. The induction of

these enzymes in green berries concurs with current understanding

that PA accumulation takes place in the early stages of berry

development [71,72]. Interestingly, ANR and LAR transcripts

(VIT_00s0361g00040, VIT_17s0000g04150) were still up-regulated

during the first ripening stage (R1) at nighttime together with the

transcription factor VvMYBPA1 (VIT_15s0046g00170), which

regulates PA synthesis [77] (Table S3). Since no further PA

synthesis is thought to take place during ripening, these results

suggest that catechin and epicatechin monomers could accumulate

in the night, while polymerization in tannosomes [73] would be

blocked. Most of the secondary metabolites synthesized by plants

are glycosylated, Williams and Harborne 1994 [74] characterized

more than 1500 glycosides of flavonoids. Ford and Hoy, 1998

identified several classes of glycosylated secondary metabolites in

grapevine berries, such as phenylpropanoids, including flavonols,

anthocyanidins, flavanones, flavones, isoflavones, and stilbenes

[75]. In this study isogenes of UDP-glycosyltransferases

(VIT_18s0001g06060, VIT_00s0324g00060, VIT_15s0046g01980,

VIT_00s1251g00010, VIT_00s0324g00050; Table S4) were in-

duced during the night in R1, which coincides with the

observations above of increased secondary metabolism. These

diurnal expression profiles could partly explain the empirical

observation that night cool temperatures are essential for the berry

quality, which is partially linked to increased contents of secondary

metabolites in grape berry skins [84].





Anthocyanin pigments are exclusively synthesized in berry skins

during ripening [76]. Expression profiles of the principal genes

involved in anthocyanin biosynthesis such as UFGT (UDPglucose:

flavonol 3-O-glucosyltransferase; VIT_04s0044g01540), VvMYBA1


and VvMYBA3 (VIT_02s0033g00450) were highly induced in

ripening berries (cluster 1 day and night; Table S3 and S5) and

thereby validate previous results obtained during day sampling on

other Vitis Vinifera varieties [75,81,82,83].

Cell Division Events Occur to a Large Extent at Night inthe Green Berry

The increase in volume and weight observed in grapevine

berries during the first growth phase is due to cell division and

expansion [4,77]. During both early development stages, up-

regulation of functional categories linked to cellular development

was observed both day and night (cell growth and death,

microtubule-driven movement, oil body organization and biogen-

esis; Figure 6). These transcriptomic changes are concomitant with

the large increase in the quantity of cell DNA observed during the

green growth stage [4]. Other authors have shown as well that cell

wall biosynthesis and cytoskeleton organization take place during

this phase, and that the related transcripts are subsequently down-

regulated in ripening berries where no major changes in the

composition of cell wall polysaccharide occurs [21,78,79].

All these categories showed noticeable diurnal variation in green

berries. The xyloglucan functional category was highly over-

represented in transcripts induced at night in G1 (Figure 6).

Several transcripts coding for xyloglucan endotransglycosylases (XET;


VIT_01s0026g00200, VIT_11s0052g01270, VIT_11s0052g01300)

were also induced at night in G1 (Table S4). Xyloglucan (XG) is a

primary cell wall hemicellulose that coats and cross-links cellulose

microfibrils. XETs can cut and rejoin XG chains, and are

therefore considered a key agent regulating cell wall expansion and

are believed to be the enzyme responsible for the incorporation of

newly synthesized XG into the wall matrix [80]. The expression

pattern of these enzymes implies an activation of cell wall

biosynthesis during the night in green berries. Several other

profiles of transcripts involved in cell wall related processes point in

the same direction. Cell division cycle protein 45 (CDC45;

VIT_12s0142g00280), which interacts in the MCM (mini-chro-

mosome maintenance) complex and plays a central role in the

regulation and elongation stages of eukaryotic chromosomal DNA

replication [81,82] was night induced in G2. In addition CDC7

(VIT_15s0021g01380, VIT_00s0616g00030), which triggers a

Figure 6. Fold change enrichment of functional categories (p,0.01) when compared to whole grapevine genome. Left part of thegraph: night down-regulated transcripts and right part of graph night up-regulated transcripts at each analyzed developmental stage.doi:10.1371/journal.pone.0088844.g006

Figure 7. Cytoscape image of day/night modulated transcripts within the phenylpropanoid pathway. Only transcripts that weremodulated at either, both green or both ripe stages (fold change.2; pval adj,0.05) are displayed. Arrow pointing upwards: up-regulated during theday (down-regulated at night); Arrow pointing downwards: down-regulated during the day (up-regulated at night). Green Arrow: Same regulation atG1 and G2; Red Arrow: Same regulation at R1 and R2; Purple Parallelogram: Day up-regulated in green stages; Day down-regulated in ripe stages;Magenta lines: Translation; Blue lines: Catalysis; Brown line: enzymatic reaction.doi:10.1371/journal.pone.0088844.g007



chain reaction resulting in the phosphorylation of the MCM

complex and ultimately in the initiation of DNA synthesis [83]

were concomitantly modulated with CENP-E-like kinetochore

proteins (VIT_13s0067g03250, VIT_13s0067g03230), a centro-

mere protein (VIT_00s0313g00010) and a putative cell elongation

protein (VIT_01s0010g01200; Table S4). Kinetochores are needed

at the onset of mitosis, where cells break down their nuclear

envelope, form a bipolar spindle and attach the chromosomes to

microtubules [84]. Indications of enhanced cell division are also

given by an up-regulation at night in G2 (Table S3) of DNA-

binding proteins (VIT_15s0048g00780, VIT_02s0025g05100) and

a DNA helicase (VIT_16s0013g00300). The transcript expression

pattern observed here confirms literature data from a molecular

point of view where cell multiplication occurs mostly in very young

berries [4]. However, to the best of our knowledge, these results

are the first on fleshy fruit demonstrating that important processes

related to cell division preferentially occur during the night.

The microtubule-driven movement functional category mainly

consists of members of the kinesin family. Kinesins are responsible

for intracellular trafficking of vesicles and organelles along

microtubules and for the dynamics of chromosomes and

microtubules in mitosis and meiosis [85,86]. These processes

seem to occur mainly in more developed green berries (G2) (cluster

3; Figure S2). In addition transcripts within this category showed

night up-regulation at G2 and curiously inversed their day/night

modulation in young green berries (G1; Table S4). Recently, it has

been proposed that kinesins intervene through transcriptional

activation activity in regulating gibberellin biosynthesis and cell

elongation [87]. This could explain the enrichment of this category

in the more advanced green berries where cell division slows down

and cell growth is more due to elongation. Since this category can

only be observed during nighttime development, it is likely that

this event has never been observed in prior transcriptomic studies

in the grapevine.

No Clear Evidence of a Pure Transcriptional Regulation ofMalic Acid Metabolism was Observed

Malic acid accumulates very rapidly during the first growth

phase and decreases throughout the second growth phase until

harvest. The switch from malic acid net accumulation to

degradation occurs at the onset of ripening [6,88,89]. Synthesis

takes place in the cytosol, through carboxylation of phosphoenol-

pyruvate (PEP) provided from glycolysis, to oxaloacetate (OAA) by

phosphoenolpyruvate carboxylase (PEPC) and further reduction

into malate (MA) by cytosolic NAD-dependent malate dehydro-

genase (NAD-MDH). Two transcripts coding for PEPCs

(VIT_01s0011g02740, VIT_12s0028g02180) were repressed follow-

ing the induction of ripening (cluster 2 day and cluster 7 night;

Table S3 and S5). This regulation matches the developmental

pattern of malate in berries. However, PEPC isogenes


were observed, exhibiting opposite expression patterns (cluster 1;

Table S5). NAD-MDH transcripts (VIT_10s0003g02500,

VIT_03s0088g01190; VIT_15s0021g02410, VIT_10s0003g01000,

VIT_10s0003g01000, VIT_01s0010g03090, VIT_19s0014g01640;

Table S5) also showed very variable patterns throughout

development. These molecular data mirror the fact that berries

can form malate from 14CO2 at any stage of development [90] and

that enzymes involved in MA synthesis are not systematically

down-regulated during ripening when no more net accumulation

of MA occurs. This observation is in accordance with the literature

where no relationship between MA content and the activities of

PEPC or malic enzyme were observed in low and high acid peach

cultivars [91], the acidless grape mutant Gora Chirine [92], apple

[93,94] and in low and high acidic loquat cultivars [95]. It

therefore seems unlikely that MA accumulation is determined by

the activity of these pathways. In plants, both the PEPC and malic

enzyme (ME) are regulated by pH in a way that contributes to the

stabilization of cytoplasm pH [25,96,97,98].

The reactions involved in malic acid breakdown are oxidation

by the Krebs cycle, gluconeogenesis, fermentation reactions that

produce ethanol, anthocyanin synthesis, and amino acid inter-

conversions [88,99,100]. Degradation takes place both in the

cytosol, by oxidation into pyruvate and PEP via malic enzyme

(ME) and phosphoenol-pyruvate-carboxykinase (PEPCK), respec-

tively, and in the mitochondria, where MA is a substrate for the

citrate cycle [101]. It should be noted that mitochondria purified

from ripening berries cannot oxidize malate in the absence of

added pyruvate, exactly as if the plant-specific mitochondrial ME

was lacking [117]. Ruffner et al. (1976) [102] reported an increase

in PEPCK activity in ripening grapes which coincides with two

PEPCK transcripts found by Terrier et al. (2005) [12]. In

microvine berries two PEPCKs were consistently up-regulated

throughout development (VIT_00s2840g00010,

VIT_07s0205g00070; cluster 8; Table S5). Together with the

observed up-regulation of MDHs (VIT_15s0021g02410,


VIT_19s0014g01640) these results confirm that the neoglucogenic

pathway via OAA (catalyzed by MDH) and PEP (catalyzed by

PEPCK) is active in the ripening berry. Functional studies on

purified membrane vesicles clearly suggest that malate metabolism

is controlled by changes affecting the bioenergetics of energy

coupling at the vacuolar membrane in fruits [89]. In Arabidopsis

thaliana, malate vacuolar transport is mediated by tonoplast

dicarboxylate transporters (TDTs) [103] and members of the

aluminum-activated malate transporter family (ALMT) [104].

AtALMT9 and AtALMT6 channels were associated with low fruit

acidity in apples [105]. In the present study, ALMT1 isogenes were

detected (VIT_08s0105g00250, VIT_09s0018g00890,

VIT_06s0009g00450, VIT_06s0009g00480; Table S5) and allocat-

ed to different clusters during the day and at night, but showed a

tendency to down-regulation during berry development. Curious-

ly, two of these isogenes (VIT_06s0009g00450,

VIT_06s0009g00480) were significantly down-regulated between

G2 and R1 at night (Table S3), whereas the others did not show

any changes between two consecutive stages. ALMT1 seems hence

not to trigger MA breakdown. By contrast, ALMT9 isoenzymes

(VIT_02s0025g00700, VIT_18s0122g00020) were induced from

G2 to R1 (Table S3). This suggests possible involvement of

ALMT9 in MA metabolism transporting it from the vacuole to the

cytoplasm to be catabolized by MDH and PEPCK.

Tartaric Acid Regulation Does not Show Significant Day/Night Variation

Tartaric acid (TA) is quantitatively the most important acid in

the mature berry [106]; as it is not used in primary metabolic

pathways after the onset of ripening, the drop in tartaric acid

concentration during ripening is due to dilution from water

import, whereas the amount of tartaric acid per berry remains

fairly constant [6,107,108]. As it is not directly affected by climatic

conditions, TA is a very important wine quality-determining

compound, in particular in warm climatic regions, and in the

context of climate warming where malic acid is consumed rapidly

resulting in a drop in total acidity and an increase in wine pH. TA

synthesis occurs in the early stages of berry development

immediately after fruit set and it levels off before the lag phase

[5]. Ascorbic acid (Asc) has been proposed as its precursor with L-

idonate dehydrogenase (L-IdnDH) showing its highest expression in



young green berries as the main rate-limiting enzyme in the TA

synthesis pathway [109]. L-IdnDH (VIT_16s0100g00290) was

down-regulated throughout berry development (cluster 7; Table

S5), matching the pattern of TA synthesis. Specific modulation at

any of the green stages was not observed which is to be expected

because L-idhDH transcripts are most abundant when TA synthesis

starts in the very early stages of development. The down-

regulation from G2 to R1 was twice as great during daytime

development as during the night. In addition L-IdhDH night up-

regulation was observed in the ripening berry without any

apparent physiological reason (Table S4).

Asc as the major precursor of tartaric acid is synthesized by the

Smirnoff-Wheeler pathway from L-galactono-1,4-lactone pro-

duced from GDP-L-mannose by the sequential action of GDP-

mannose-3,5-epimerase (GME), GDP-L-galactose phosphorylase (VTC2), L-

galactose-1-phosphate phosphatase and L-galactose dehydrogenase (L-

GalDH), the direct ascorbate precursor [110]. Galacturonic acid

from cell walls was suggested as an alternative major precursor of

ascorbate with galacturonate reductase as a possible regulatory step

enzyme [111]. Three VTC2 isoenzymes were detected of which

two (VIT_14s0006g01370, VIT_10s0003g05000) were slightly up-

regulated throughout berry development (cluster 1 and cluster 7;

Table S5). Only one (VIT_19s0090g01000; cluster 2) was down-

regulated as expected given its putative role in TA synthesis, which

ceases just before the lag phase.

Day Seems to be as Important as Night in Amino AcidMetabolism

Free amino acids and ammonia make up the majority of

nitrogen (N) containing compounds. Half of the berry’s total

nitrogen is imported during ripening where proline (pro) and

arginine (arg) account for over 70%. Only a-amino acids (pro is

not fermented) are important yeast nutrients and thus needed for

successful alcoholic fermentation [112,113]. In addition they

contribute to a considerable extent to varietal flavor in the finished

wine [114].

In this study, most analyzed amino acids exhibited a steady

increase from fruit set throughout ripening (Table S1.) Only

glutamine (gln) was accumulated very early and steadily from

berry set (BS) to G2 and thereafter decreased slightly from R1 to

R2. Gln is a nitrogen donor for many biosynthetic reactions,

including the biosynthesis of other amino acids, purines, pyrim-

idines, glucosamime and carbamoyl phosphate and its biosynthesis

is catalyzed by glutamine synthetase. Consistently glutamine

synthetase isogenes (VIT_16s0100g00580, VIT_03s0088g00570,

VIT_05s0020g02480; Table S3) were highly up-regulated at G2

and three other isogenes were induced from young to ripening

stages (VIT_07s0104g00170, VIT_08s0007g04670,

VIT_10s0042g01000; Table S5).

A transcript coding for NADH glutamate synthase

(VIT_07s0005g00530) which catalyzes the reaction from gln to

glutamate (glu) was down-regulated (cluster 2, day and night) in

ripening berries in addition to GLT1 (NADH-dependent glutamate

synthase 1) genes (VIT_16s0098g00290, VIT_15s0024g01030),

where the second transcript was only detected during daytime

development. The complex regulation of glu and gln does not

permit any conclusive statement to be made about the molecular

events occurring during berry development during the day and at

night.

In grapevine berries, pro accumulation starts very late during

the first growth phase and continues throughout ripening [115],

arg, the other principal amino acid, which shares significant

pathway features with pro, begins to accumulate earlier in the

green berry and continues during ripening. Arg accumulation

levels off early during ripening in cultivars exhibiting very high pro

concentrations [116], which, on the basis of this study, also seems

to be true for the microvine. Arg was present in green berries, but

a significant increase was observed both in pro and arg, in

particular in ripening berries. There are two pathways of pro

biosynthesis in higher plants. The first is from glu, which is

converted to pro by two successive reductions catalyzed by

pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reduc-

tase (P5CR), respectively. P5CS is a bifunctional enzyme catalyzing

firstly the activation of glu by phosphorylation and secondly the

reduction of the labile intermediate c-glutamyl phosphate into

glutamate-semialdehyde (GSA), which is in equilibrium with the

P5C form [117,118]. Although it has been shown that pro

accumulation in grapes occurred independently from P5CS which

was expressed evenly during berry development and in which

other regulation systems probably intervene [115], we detected

P5CS isogenes (VIT_15s0024g00720, VIT_08s0007g01060), which

were up-regulated in ripening berries (cluster 1; Table S5) where

pro is accumulated. This is in agreement with other microarray

studies carried out on Cabernet Sauvignon [18] and Trincadeira

[17]. Moreover, three pro transporter isogenes were detected

(VIT_13s0019g03220, VIT_13s0073g00290, VIT_07s0141g00640;

Table S3) and correlated with pro accumulation during up-

regulation from G2 to R1 without showing any day/night

specificities.

An alternative pathway starts with the pro precursor ornithine,

which can be transaminated to P5C by ornithine aminotransferase

(OAT), a mitochondrial-located enzyme [119]. An OAT tran-

script (VIT_10s0003g03870) was down-regulated in G1 (cluster 6;

Table S5) during the day, suggesting that this pathway may not be

important in green berries. A glutamate decarboxylase (GDC)

transcript (VIT_01s0011g06610) producing c-aminobutyrate was

induced in green berries before the lag phase, and then

continuously down-regulated (cluster 7 day and night; Table S5).

The latter transcript exhibited as well a day induction in G2. As c-

aminobutyrate is also a stress marker this could explain the

daytime up-regulation in response to higher day temperatures in

green berries.

Lysine-histidine transporters (LHT) show a very high affinity for

amino acids, and LHT1 in particular belongs to a class of amino

acid transporters that is specific for lys and his [120]. It has been

shown that LHT1 is involved in the uptake of amino acids from

soil into the leaf mesophyll cells [121]. No clear pattern in LHT1

isogenes was observed in this study: Some isogenes


were up-regulated in G1 (cluster 5 day and night; Table S5)

whereas others (VIT_06s0061g01210, VIT_14s0171g00400, cluster

8 day and 9 night; Table S5) showed opposite patterns.

Genes Involved in Terpene and Carotenoid BiosynthesisShow Circadian Patterns

Terpenoid volatiles, principally monoterpene alcohols such as

linalool, geraniol, nerol and terpineol are important flavor and

aroma compounds of grapevine berries and wine, and most

accumulate during ripening [122,123]. For example, in fruits of

the cultivar Muscat, the terpenoid content paralleled sugar

accumulation and several monoterpenes reached peak levels in

the overripe fruit [124], though present molecular data does not

unambiguously confirm this. Monoterpenes are products of the

isoprenoid pathway from the intermediates isopentenyl-pyrophos-

phate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP).

IPP is synthesized via the non-mevalonate pathway that requires

1-deoxy-D-xylulose 5-phosphate synthase. The transcript coding for this

enzyme (VIT_09s0002g02050) was consistently down-regulated



during berry development (cluster 7 day; cluster 2 night; Table S5)

whereas isopentenyl diphosphate isomerase 2 transcripts, catalyzing the

conversion of IPP to DMAPP were induced in ripening berries


cluster 1 day and night; Table S3 and S5).

Geraniol 10-hydroxylase (G10H) is thought to play an

important role in iridoid monoterpenoid and indole alkaloid

biosynthesis [125]. Most G10H transcripts were induced in

ripening berries to the same degree at day and night (cluster 1;

Table S5). However, two transcripts (VIT_02s0012g02370,

VIT_02s0012g02380) showed nighttime induction in ripening

berries, which was most pronounced at the latest stage (Table

S4). Several transcripts coding for the enzymes involved in the

biosynthesis of the bicyclic monoterpene pinene were found to be

modulated. Pinene has a woody-green pine aroma and is one of

the most widely detected volatile organic compounds emitted by

plant into the atmosphere [126]. Several homologues of pinene

synthase showed down-regulation in ripening berries (Table S3 and

S5). Two of the transcripts (VIT_08s0007g06860,

VIT_12s0059g02710) were induced at night in R1 (Table S4).

The tendency to exhibit a circadian expression pattern of pinene

synthase-coding transcripts has been observed in Artemisia annua

[126], but here this day/night pattern was observed at only one

berry developmental stage.

Two sesquiterpene synthases, (+)-valencene- and (2)-germa-

crene D-synthase have been recently characterized in Vitis Vinifera

L. berries. Their expression was principally induced during later

stages of berry development, several weeks after the onset of

ripening [127]. Consistent with this, it was found that a valencene

synthase (VIT_18s0001g04050) and a (2)-germacrene D synthase were

induced in ripening berries (Table S3). Several isogenes of (2)-

germacrene D synthase exhibited night up-regulation in R1


VIT_18s0001g05240; Table S4) suggesting a circadian regulation

amongst genes in terpene biosynthesis.

An important subgroup of terpenes are carotenoids, a hetero-

geneous group of plant isoprenoids primarily present in the

photosynthetic membranes of all plants where they quench triplet

chlorophyll, singlet oxygen, and also superoxide anion radicals

[128]. The first committed step in carotenoid biosynthesis is the

production of the 40-carbon phytoene from condensation of two

geranylgeranyl pyrophosphate (GGPP) molecules, catalyzed by the

enzyme phytoene synthase (PSY). Three PSYs were detected

showing opposite expressions hence not presenting a consistent

pattern during berry development (Table S5).

The cleavage of carotenoids can lead to the formation of C13-

norisoprenoids and the phytohormones abscisic acid and strigo-

lactone. C13-norisoprenoids are important flavor compounds

contributing to varietal character of grapes and wine. In the

grapevine, a direct relationship between a decrease in carotenoid

concentration and C13-norisoprenoid production has been dem-

onstrated [129]. The C13-norisoprenoids identified in wine with

important sensory properties are TCH (2,2,6-trimethylcyclohex-

anone), b-damascenone, b-ionone, vitispirane, actinidiol, TDN

(1,1,6-trimethyl-1,2-dihydronaphthalene), riesling acetal and TPB

(4-(2,3,6-trimethylphenyl)buta-1,3-diene) [130]. The principal

enzyme involved in the cleavage of carotenoids to C13 norisopre-

noids is carotenoid cleavage dioxygenase 1 (CCD1), which has been

characterized in grapes where it exhibited an induction of gene

expression towards ripening [131]. In the present study a putative

CCD1 homologue (VIT_02s0087g00930) was identified that was

highly up-regulated towards ripening (cluster 1 day and night;

Table S3 and S5) supporting the results obtained by Mattieu et al.,

2005 [131] where C13-norisprenoid synthesis takes place rather in

ripening berries occurring after CCD induction.

Circadian Clock Related Transcripts Followed Day/NightPatterns Mainly in Green Berries

The circadian clock consists of morning, core, and evening

interlocking feedback loops [132]. The MYB transcription factors

CCA1 (circadian clock associated1) and LHY (late elongated hypocotyl)

belong to the core loop in Arabidopsis thaliana [29]. CCA1 regulates

homeostasis of ROS (reactive oxygen species) and would thus

coordinate time-dependent responses to oxidative stress [133]. In

both green stages, a CAA1 transcript (VIT_15s0048g02410; Table

S4) was considerably induced at night while LHY responded only

in G1. CIR1, a third circadian clock-related transcript putatively

involved to the core loop (VIT_04s0079g00410; Table S4) was

found to be day/night modulated at all stages but R2. The

morning loop induces PRR9 and PRR7 (pseudo response regulator)

that are linked to CCA1/LHY [134,135]. In microvine berries

isogenes of PRR7 (VIT_13s0067g03390, VIT_06s0004g03660,

VIT_06s0004g03650), PRR9 (VIT_15s0048g02540) and a PRR5

(VIT_16s0098g00900) were concomitantly induced during the day

but only in the first green stage of berry development (Table S4). A

putative GI (gigantea) transcript (VIT_18s0157g00020) identified in

the evening loop [136] and epistatic to ELF4 (early flowering 4) [137]

was down-regulated at G1 whereas ELF4 (VIT_13s0067g00860)

showed night induction at both G1 and G2. A homologue

(VIT_07s0104g00350) to ZGT acting as a coupling agent between

the central circadian oscillator and rhythmic LHCB1 (light harvesting

complex) was induced during the day in G1 and G2. It may be

concluded that green berries are significantly more responsive to

the circadian cycle than ripe berries. It can be hypothesized that

this is due to the fact that ripe berries have reserves in the form of

fructose and glucose, whereas green berries photosynthesize

during the day and many genes associated with the circadian

clock are somehow involved in photosynthesis also.

Heat Shock Related Genes and Transcription FactorsChange their Day/Night Expression Pattern According toDevelopmental Stage

The multi-protein-bridging factor 1c (MBF1c) previously character-

ized in Arabidopsis thaliana functions upstream to salicylic acid,

ethylene and trehalose upon heat stress [138,139]. In microvine

berries, MBF1c showed consistent up-regulation towards ripening

(VIT_11s0016g04080; cluster 6 day and cluster 8 night; Table S5).

This heat shock responsive transcription factor would be expected

to be daytime induced as well due to the temperature gradient

between day and night (DTday +10uC). MBF1c was induced during

the day in green berries, but no modulation was observed in ripe

berries, indicating a higher temperature sensitivity of the green

berry.

VvGOLS1 (galactinol synthase) has recently been identified as being

temperature regulated in berries of Cabernet Sauvignon L. [140].

This gene is transactivated by the heat shock transcription factor

VvHSFA2 [140]. In microvine berries, several galactinol synthase

coding isogenes were modulated throughout berry development

and/or during the day/night (Table S3–S5). Ten of these probe

sets exhibited day/night co-regulation - all were day up-regulated

in green berries and inversely modulated in ripening berries.

However, they did not show a common pattern of regulation

throughout berry development. The VvGOLS1 gene locus

(VIT_07s0005g01970) from Pillet et al., 2012 [141] showed

consistent up-regulation throughout development (cluster 6 day

and cluster 8 night) and day induction only at G2. As the day/



night temperature gradient was +10uC it was expected that

VvGOLS1 would be activated during the day as it is very responsive

to heat stress. However, in ripening berries, it seemed to loose this

function like MBF1c: circadian changes appeared thus to have

greater impact than the day/night temperature gradient.

In plants, bHLH (basic helix-loop-helix) proteins function as

transcriptional regulators modulating secondary metabolism, fruit

dehiscence, carpel and epidermal development, phytochrome

signaling, and responses to environmental factors [141,142,143].

This functional category showed continuous down-regulation

throughout berry development, with a peak in the young green

berry where major events in early reproductive development occur

(cluster 7, Figure S3). Furthermore, enrichment could be observed

only during night development, confirming the previous hypoth-

esis that significant changes in cellular division take place at night

in green berries, as supported by the expression pattern of a

transcript coding for SPATULA (VIT_18s0001g10270) which

affected cell proliferation in Arabidopsis thaliana [144].

EthyleneAs the grapevine fruit ripens without ethylene and does not

exhibit a respiration burst nor high production of ethylene it has

consequently been classified as non-climacteric [145]. However,

Chervin et al., 2004 [146] reported a modest increase in ethylene

at the onset of ripening in the grapevine. The same authors

observed a correlation between ethylene accumulation and the

expression of 1-aminocyclopropane-1-carboxylate oxidase (AOC) tran-

scripts and enzyme activity in berries. AOC catalyzes the final

reaction step from ACC to ethylene [147] and has been also

identified in the wall of apple and tomato fruit cells [148]. Eleven

AOC isogenes were detected without exhibiting a common pattern

throughout development. However ethylene receptor coding

transcripts (ETR1; VIT_19s0093g00580, ETR2;

VIT_06s0004g05240) were induced during development in ripen-

ing berries (ETR1: cluster 1 night, cluster 6 day and ETR2: cluster

1 night, cluster 8 day; Table S5). In addition to these

developmental regulations, ETR2 showed nighttime induction in

ripening berries (Table S5). These results support the hypothesis of

ethylene intervention in berry ripening whose role might be in

relation to berry architecture or anthocyanin accumulation

[146,149]. Taking this into account, together with the observed

abundance of principal phenylpropanoid pathway transcripts at

night in ripe berries, putative involvement of ethylene in secondary

metabolism could be supposed. However, indications do exist that

the circadian rhythm plays a critical role in ethylene regulation

and should be taken into account in further hormonal studies.

Abscisic AcidAbscisic acid (ABA) intervenes in embryo and endosperm

formation during seed development, in seed dormancy in mature

berries and has a promotive role during fruit ripening [98].

Highest ABA levels are found in very young berries, which then

decrease until ripening, where accumulation resumes in parallel

with coloration and sugar accumulation [145,150]. The rate

limiting enzyme in ABA synthesis, 9-cis-epoxycarotenoid dioxygenase

[151] (NCED; VIT_02s0087g00930), steadily increased throughout

berry development (cluster 5; Table S5), which is in agreement

with previous studies on other varieties [18]. Another important

enzyme involved in ABA synthesis is zeaxanthin epoxidase (ZEP),

which catalyzes zeaxanthine biosynthesis, a carotenoid precursor

for ABA [152]. There are few data on ZEP available - Deluc, et al.

2007 [18] observed a steady decrease in expression in Cabernet

Sauvignon L. during berry development, the same pattern of ZEP

transcripts (VIT_00s0533g00020; VIT_13s0156g00350,

VIT_07s0031g00620; cluster 2 night, cluster 7 day; Table S5)

was found in microvine berries at day and night.

An NCED transcript was found to be induced during the day in

green berries but this expression was inversed in R1

(VIT_19s0093g00550; Table S4). In Arabidopsis thaliana induction

of this enzyme led to greater stress tolerance to intense light and

high temperatures [153]. CYP707A1 (VIT_02s0087g00710) and

CYP707A2 (VIT_07s0031g00690) encode for abscisic acid 8’-

hydroxylases which controls seed dormancy and germination in

Arabidopsis thaliana [154]. Interestingly, they also exhibited night-

time up-regulation of CYP707A1 at all stages but in young green

berries CYP707A2 was induced only in G2 (Table S4). Generally

ABA also plays a role in abiotic and biotic stress tolerance in plants

[155], thus these results reinforce the observation that oxidative

stress appears to occur during the night in ripening berries.

However, the opposite was observed in regards to the ABA-

mediated signaling category, which was significantly enriched in

transcripts down-regulated at night in R1 (Figure 6). This was

mainly due to isogenes of ATHVA22A (Arabidopsis thaliana HVA22

homologue A) that were up-regulated during the day in R1 and in R2

(Table S4). HVA22 is mediated by ABA and was induced by cold

and drought stress in barley [156]. It has been shown that HVA22

is a ER- and golgi-localized protein that negatively regulates GA-

mediated vacuolation and programmed cell death [157]. This

regulation pattern cannot be explained by temperature neither by

the previously described oxidative stress hypothesis occurring at

night in ripening berries. Nonetheless, it shows though that the

genes of this family appear to be moderately responsive to diurnal

and developmental changes.

GibberellinsGibberellins (GAs) are regulators of many plant development

processes, mainly cell division and expansion. During the

reproductive development of the grapevine, GAs are known to

be involved in the regulation of grapevine fruit set and young berry

expansion. Accordingly, GA levels during berry development are

high around flowering and early in berry development and

decrease steadily thereafter [158]. Two gibberellin receptor coding

transcripts (GID1L3; VIT_15s0048g01390, VIT_15s0048g01350)

were night up-regulated in R2 (Table S4). Similar night induction

in ripening berries was observed in relation to Gibberellin oxidases

(GA 20ox2: VIT_03s0063g01290, VIT_03s0063g01280 and GA 2ox:

VIT_05s0077g00520), enzymes involved in GA metabolism in

higher plants [159]. During berry development many isogenes

coding for the above enzymes where allocated to different clusters

exhibiting no clear expression pattern (Table S5). No conclusions

can be drawn regarding GA developmental regulation; day/night

expression patterns of detected transcripts indicate their putative

involvement in secondary metabolism, which was found to be

highly active at night in ripening berries.

CytokininsCytokinins intervene in the establishment of the vasculature

during embryonic development; they control the number of early

cell divisions and have a regulatory control on meristem activity

and organ growth during postembryonic development [160]. In

the grapevine berry they are thought to be involved in fruit set and

growth promotion with maximum concentrations in young

berries, decreasing towards ripening. [161]. Induction of tran-

scripts was observed in young green berries, which are involved in

mediating cytokinin reception and transport, such as histidine kinase

(AHK4/WOL; VIT_01s0011g06190) acting as a cytokinin receptor

protein [162], (cluster 5 day; Table S5). Purine permease 1 (PUP1;

VIT_18s0001g06950, VIT_18s0001g06940, VIT_18s0001g06910),



involved in cytokinin transport [163] showed consistent up-

regulation throughout berry development (cluster 6 day and

cluster 8 night). Isopentenyltransferase, catalyzing the rate-limiting

step in cytokinin biosynthesis in Arabidopsis thaliana [164]

(VIT_09s0070g00710, VIT_07s0104g00270) was concomitantly

regulated (cluster 6 day and night; Table S5) and exhibited

additional up-regulation during the day in R1 (Table S4). It was

not possible to confirm the results of Deluc et al., 2011 [18] who

observed a steady decrease in a putative cytokinin oxidase during

berry development, probably related to decreases in cytokinin

content. In microvine berries three transcripts coding for a

putative cytokinin oxidase (VIT_00s2520g00010,

VIT_00s2191g00010, VIT_00s0252g00040) were strongly up-

regulated (cluster 1; Table S5) in ripening berries, indicating that

this enzyme probably does not play a major role in cytokinin

synthesis. Many cytokinin-mediated transcripts were down-regu-

lated at night in G1 (see functional category cytokinin-mediated

signaling in Figure 6). Most of these probesets were homologues to

the pseudo-regulators (PRRs) that were discussed above in the

circadian clock section.

ConclusionTo our knowledge this is the first genome-wide transcriptomic

study on fleshy fruits deciphering night regulations throughout

development, and comparing day/night gene expression changes

at different stages. All developmentally regulated transcripts

detected during the day were also detected at night, validating

previous approaches based solely on day sampling. Day expression

data was well correlated with other expression data obtained on a

non-dwarf genotype grown in the field.

Here, advantage has been taken of the microvine model to

perform simultaneous sampling of fruits at several developmental

stages from the same plant. Due to the size of the microvine,

experiments could be performed in climatic chambers under

strictly controlled environmental conditions (i.e. day/night radi-

ation, temperature, vapor pressure deficit) unprecedented in other

development studies on grapevine fruit development. Thereby

experimental noise, affecting gene expression in a non-quantifiable

way, was reduced to a minimum. It was demonstrated that 20% of

developmentally-regulated transcripts were only detected during

the night and that very few transcripts are day/night regulated

consistently throughout all stages of development. This indicates

that photoperiod regulation drastically changes at the onset of

sugar storage in berries. In many pathways, it was observed that

the gene expression pattern showed a day/night variation with

changes in relation to sampling stage. This is particularly

noticeable with respect to cell wall-related processes that are more

active during night in the young fruit. Significant observations

were made in relation to secondary metabolism-related enzymes

that were only present in the ripening berry during the night.

Several processes showed an inversion of their day/night

regulation between green and ripe berries, such as sugar transport

and phytoalexin synthesis, which were more pronounced during

the day in green berries and vice versa in ripening berries.

Interestingly, the oxidative burst transiently detected by several

authors at the onset of ripening was observed to occur at nighttime

in the ripening berry.

For a greater understanding of the mechanisms involved in the

regulation of berry development, it appears to be essential to

evaluate different processes and events both during the day and at

night. Considering the significant diurnal changes observed during

this study on plants grown under controlled conditions, it would

also seem necessary to investigate the transcriptomic response to

abiotic stresses and its day – night modulation at different stages of

development.

Materials and Methods

Plant MaterialOne year old own-rooted microvines were grown in a

greenhouse until a stable fructification was established. The

reproductive system was normalized among all plants by removing

organs up to flowering. Plants were further grown in climatic

chambers (2 m2). One whole developmental cycle was undergone

under fully controlled conditions (day/night temperature: 30/

20uC, Photoperiod: 14 h, VPD: 1 kPa). Reproductive organs were

sampled in biological triplicates two hours before the end of the

day and the end of the night and were immediately frozen in liquid

N2. 30 berries per replicate were crushed into liquid N2 and the

obtained powder was used for biochemical analysis and RNA

extraction.

Organic Acid and Sugar AnalysisFor organic acid, glucose and fructose approximately 0.1 g of

powder was diluted five fold in deionized water and samples were

frozen at 220uC. Prior to analysis diluted aliquots were defrosted

and subsequently heated (60uC for 30 min). After cooling to

ambient temperature, samples were homogenized and diluted with

4.375 mM acetate as an internal standard. To avoid potassium

bitartrate precipitation, 1 mL sample was mixed with 0.18 g of

Sigma AmberliteH IR-120 Plus (sodium form) and agitated in a

rotary shaker for at least 10 hours before centrifugation

(13000 rpm for 10 min). The supernatant was transferred into

HPLC vials before injection on Aminex HPXH87H column eluted

in isocratic conditions (0.05 mL.min21, 60uC, H2SO4) [165].

Organic acids were detected at 210 nm with a waters 2487 dual

absorbance detectorH. A refractive index detector Kontron 475Hwas used to determine fructose and glucose concentration.

Concentrations were calculated according to Eyegghe-Bickong

et al. 2012 [166].

Amino Acid AnalysisPrimary amino acids were analyzed using a modified version of

a previously reported method [167]. A Hewlett-Packard (Agilent

Technologies Massy, FranceH) 1100 179 series HPLC instrument

was used, with a G1321A fluorescence detector set at excitation

and emission wavelengths of 330 nm and 440 nm, respectively.

Separations were carried out on a 150 mm63 mm Macherey

Nagel DurabondH column 5 mm dp, protected by a 1 mm C18

SecurityGuardH cartridge supplied by Phenomenex (France).

Mobile phase A consisted of 95% 0.05 M acetate buffer, pH 6.5

and 5% methanol:acetonitrile [1:1] filtered under vacuum using a

0.22 mm nylon membrane. Mobile phase B consisted of

methanol:acetonitrile [1:1]. Separations were carried out at

40uC with a flow rate of 0.5 ml/min. As proline does not react

with OPA, a new high-throughput spectrophotometric method

was developed and validated for its analysis. Briefly, the method

involves reacting the sample with ninhydrin in DMSO and formic

acid at 100uC for 15 minutes to yield a salmon pink reaction

product. Under these conditions, primary amino acids do not react

with ninhydrin and thanks to the particular solvent composition,

the extraction and centrifugation steps reported in similar methods

are avoided.

RNA ExtractionRNA extraction was carried out using an in-house extraction

buffer containing 6 M guandine-hydrochloride, 0.15 M tri-sodi-



um-citrate, 20 mM EDTA and 1.5% CTAB. Five volumes of

room temperature extraction buffer supplemented with 1% MSH

were added to 1 g of powder followed by immediate agitation. Cell

debris was removed by centrifugation, and after chloroform

treatment one volume isopropanol was added to precipitate RNA.

Samples were kept at –20uC for at least two hours. RNA was

precipitated by centrifugation washed with 75% ethanol and the

pellet was suspended with RLC Buffer from the Quiagen

rnaEasyH Kit previously supplemented with 1.5% CTAB. To

reduce pectin and tannin residues an additional chloroform

treatment was carried out. The succeeding washing steps and the

DNAase treatment are performed as described in the kit.

Absorbance was measured at 260 and 280 nm and the concen-

tration of RNA was determined with a NanoDrop 2000c

Spectrophotometer (Thermo ScientificH). The integrity of RNA

was evaluated using an 2100 Bioanalyzer (Agilent TechnolgiesH).

Nimblegen 12x Microarray HybridizationcDNA synthesis, labeling, hybridization and washing reactions

were performed according to the NimbleGen Arrays User’s Guide

(V 3.2). Hybridization was performed on a NimbleGen microarray

090818 Vitis exp HX12 (Roche, NimbleGen Inc., Madison, WI),

consisting of 29,549 predicted genes on the basis of the 12X

grapevine V1 gene prediction version V1 http://srs.ebi.ac.uk/.

The chip probe design is available at the following url: http://

ddlab.sci.univr.it/FunctionalGenomics/. The raw data is available

at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/

geo/info/linking.html) under the series entry GSE52829.

Statistical AnalysisThe Robust Multi-array Analysis (RMA) algorithm was used for

background correction, normalization and expression levels [168].

Differential expression analysis was performed with the bayes t-

statistics from the linear models for microarray data (limma) [169].

P-values were corrected for multiple-testing using the Benjamini-

Hochberg’s method [170]. Transcripts were considered as

significantly modulated when absolute change was .2 fold (log2

fold change .1) and adjusted p. value was ,0.05 between two

conditions. Gene clustering was performed on mean centered

values of RMA normalized and log2 transformed expression data.

This analysis was performed using the Multiple Experiment

Viewer version 4.6.2H software package, and based on the k-

means method using Pearson’s correlation distance calculated on

the gene expression profiles. Gene annotation was derived from

Grimplet et al., 2012 [171].

Visualization of Grapevine Transcriptomics Data UsingMapMan Software

Information from the Nimblegen microarray platform was

integrated using MapMan software [172] as described for the

Array Ready Oligo Set Vitis Vinifera (grape), V1.0 (Operon,

Qiagen), and the Affymertix GeneChipH Vitis Vinifera Genome

Array [64] (correspondence from Grimplet et al.; 2012 [172].

Mapman pathway analysis was performed with day and night-

specific transcripts allocated to cluster 1 and 2, respectively. For

identified genes, the fold change between G2 and R1 was

calculated and mapped on the pathway ‘‘metabolism overview’’.

Day-specific values were mapped in red and night-specific ones in

blue.

Cytoscape Pathway AnalysisFor the illustration of the phenylpropanoid pathway, transcripts

that were significantly and concomitantly modulated (fc .2, p,

0.05) in either both green or both ripe stages were mapped using

VitisNet networks through cytoscape v 2.8.3 s [173].

Functional CategoriesTranscripts allocated to day - night development clusters or

identified by statistical testing were analyzed with FatiGO [174] in

order to identify significant enrichment of functional category.

Categories were derived form [171] and Fisher’s exact test was

carried out to compare genes list with non-redundant transcripts

from the grapevine genome. Significant enrichment was consid-

ered in case of p value ,0.01 and illustrated as fold change.

Supporting Information

Figure S1 Fold change of enriched functional categoriesof transcripts allocated to cluster 1 and 2. Categories for all

day and night as well as for day and night specific transcript within

cluster is illustrated.

(PDF)




(PDF)




(PDF)




(PDF)

Figure S5 Correlation between genes expression (log2)between green and ripening stages of Corvina L. (Fasoliet al., 2012) and microvine berries.

(BMP)

Table S1 Amino acid content of sampled berries.

(XLSX)

Table S2 Overview of the number of up and down-regulated

transcripts within all developmental stages.

(XLSX)

Table S3 All modulated transcripts between developmental

stages.

(XLSX)

Table S4 Day – Night modulated transcripts.

(XLSX)

Table S5 Transcripts allocated to clusters.

(XLSX)

Table S6 Transcripts, identified in Corvina L. as well as in

microvine berries between green and ripe stages.

(XLSX)

Acknowledgments

For technical support during climatic chamber experiments, support

during sampling and with sample processing, we would like to thank

Rattaphon Chatbanyong, Gilbert Lopez, Marc Farnos, Clea Houel, Agnes

Ageorges, Therese Marlin, Sandrine Vialet and Bertrand Muller.



https://www.researchgate.net/publication/224895617_Comparative_analysis_of_grapevine_whole-genome_predictions_functional_annotation_and_categorization_of_the_predicted_gene_sequences?el=1_x_8&enrichId=rgreq-f1a1c3cc-85d5-40bc-b7a7-5dc03bfa8b14&enrichSource=Y292ZXJQYWdlOzI2MDI1NDc5NDtBUzoxMDQ2MTcyNTc5OTYyODlAMTQwMTk1NDEwNjA5OA==

Author Contributions

Conceived and designed the experiments: MR CR. Performed the

experiments: MR CR NL AP LT. Analyzed the data: MR JG. Contributed

reagents/materials/analysis tools: MR CR MK LT. Wrote the paper: MR

CR. Paper corrections: MK LT JG. Administrative supervision: LT. Plant

culture: NL LT AP.

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Is Transcriptomic Regulation of Berry Development More Important at Night than During the Day?

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